Expansion and long-term culture of differentiated normal rat urothelial cells in vitro

The objective of this study is to establish a reliable cell culture system for the long-term culture of rat urothelial cells (RUC), in which the cells multiply in vitro and form stratified polarized urothelium. Urothelial cel ls were harvested by the enzymatic digestion of the urothelium exposed by t he eversion of resected rat bladders. Primary cultures were initiated in ke ratinocyte serum-free medium (KSFM) for selective proliferation of urotheli al cells. Subsequently, the cells were propagated in a mixture of condition ed medium (CM) derived from Swiss 3T3 cell culture. supernatant and KSFM CM -KSFM). Mean population doubling time was 13.8 +/- 0.9 h. RUC were successf ully maintained for 18 passages over a period of 4-5 mo. Detailed investiga tions of culture conditions showed that CM-KSFM yielded a differentiated mu ltilayer structure. The stratified urothelial sheets measuring 4 X 6 cm(2) could be formed and (lien detached using dispase. Cytokeratin pattern in bo th the cultured urothelial monolayer and engineered stratified layers was s imilar to those seen in vivo, as assessed with monoclonal antibody against cytokeratin 17. Ultrastructural morphology showed microvilli. basal cell la yer, and desmosomes between adjacent cells in the stratified urothelium.